Genetic analysis of the viral genes that contribute to the immortalization of human B-lymphocytes by Epstein-Barr virus (EBV) has been hampered by the lack of a reliable means for testing mutant viruses in the absence of contaminating wild-type virus. Dr. Sugden, in collaboration with his longstanding colleague, Dr. Hammerschmidt have developed derivatives of EBV, termed mini-EBVs, that can be propagated as plasmids in E. coli, packaged in a human cell line containing an EBV helper virus, and are capable alone of immortalizing B-lymphocytes. Defined mutations can be introduced into mini-EBVs in their E. coli host. Drs.Sugden and Hammerschmidt propose to measure the effects of defined mutations in selected EBV immortalizing genes on the efficiency of immortalization. These genes will also be analyzed to determine whether they are required for the initiation and/or the maintenance of immortalization. Finally, mini EBVs containing the minimal amount of genetic information required for immortalization will be constructed.